Corn event PV-ZMIR13 (MON863) plants and compositions and methods for detection thereof

ABSTRACT

The present invention provides compositions and methods for detecting the presence of the corn event MON863 DNA inserted into the corn genome from the transformation of the recombinant construct containing a Cry3Bb gene and of genomic sequences flanking the insertion site. The present invention also provides the corn event MON863 plants, progeny and seeds thereof that contain the corn event MON863 DNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35USC§371 application of PCT/US03/22860, filedJul. 23, 2003, which claims the benefit of priority to U.S. ProvisionalApplication 60/399,279, filed Jul. 29, 2002.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology.The invention more specifically relates to a coleopteran resistant cornplant (Zea mays) PV-ZMIR13, designated MON863, and to seeds and progenyof the corn plant MON863. The corn plant MON863 and its progeny areparticularly resistant to Diabrotica vergifera, Diabroticaundecimpunctata, and Leptinotarsa decemlineata.

The present invention more specifically also relates to a DNA constructinserted into the corn plant genome in event MON863 for conferringresistance to insect infestation by a coleopteran species. The presentinvention also relates to assays for detecting the presence of a cornplant MON863 DNA in a sample and compositions thereof.

BACKGROUND OF THE INVENTION

Corn is an important crop and is a primary food source in many areas ofthe world. The methods of biotechnology have been applied to corn plantsfor improvement of the agronomic traits and the quality of the product.Expression of foreign genes in plants is known to be influenced by theirchromosomal position, perhaps due to chromatin structures (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., Ann.Rev. Genet 22:421-477, 1988). For this reason, it is often necessary toscreen a large number of events in order to identify an eventcharacterized by optimal expression of an introduced gene of interest.For example, it has been observed in plants and in other organisms thatthere may be a wide variation in levels of expression of an introducedgene among events. There may also be differences in spatial or temporalpatterns of expression, for example, differences in the relativeexpression of a transgene in various plant tissues, that may notcorrespond to the patterns expected from transcriptional regulatoryelements present in the introduced gene construct. For this reason, itis common to produce hundreds to thousands of different events andscreen those events for a single event that has desired transgeneexpression levels and patterns for commercial purposes. An event thathas desired levels or patterns of transgene expression is useful forintrogressing the transgene into other genetic backgrounds by sexualoutcrossing using conventional breeding methods. Progeny of such crossesmaintain the transgene expression characteristics of the originaltransformant. This strategy is used to ensure reliable gene expressionin a number of varieties that are well adapted to local growingconditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the premarket approval and labeling of food derived fromrecombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well-known nucleic acid detection methodsuch as the polymerase chain reaction (PCR) or DNA hybridization usingnucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, terminators, markergenes, etc. As a result, such methods may not be useful fordiscriminating between different events, particularly those producedusing the same DNA construct unless the sequence of chromosomal DNAadjacent to the inserted DNA (“flanking DNA”) is known. Anevent-specific PCR assay is discussed, for example, by Windels et al.(Med. Fac. Landbouww, Univ. Gent 64/5b: 459-462, 1999), who identifiedglyphosate tolerant soybean event 40-3-2 by PCR using a primer setspanning the junction between the insert and flanking DNA, specificallyone primer that included sequence from the insert and a second primerthat included sequence from flanking DNA.

SUMMARY OF THE INVENTION

According to one preferred embodiment of the present invention,compositions and methods are provided for detecting the presence of thetransgene/genomic insertion region from a novel corn plant PV-ZMIR13,designated MON863. DNA sequences are provided that comprise at least onejunction sequence of MON863 selected from the group consisting of SEQ IDNO:1 (arbitrarily assigned 5′ end insert-to-genome junction) and SEQ IDNO:2 (arbitrarily assigned 3′ end insert-to-genome junction) andcomplements thereof, wherein the junction sequence spans the junctionbetween a heterologous DNA inserted into the corn genome and the DNAfrom the corn cell flanking the insertion site and is diagnostic for theevent.

According to another preferred embodiment of the present invention, DNAsequences that comprise the novel transgene/genomic insertion region,SEQ ID NO:3 (sequence containing the arbitrarily assigned 5′ end of theinserted DNA) and SEQ ID NO:4 (sequence containing the arbitrarilyassigned 3′ end of the inserted DNA) for example, are disclosed.

According to still another preferred embodiment of the presentinvention, the DNA sequences that comprise at least from about 11 toabout 50 or more nucleotides of the 5′ transgene portion of the DNAsequence of SEQ ID NO:7 and a similar length of 5′ flanking corn DNAsequence of SEQ ID NO:5, or a similar length of 3′ transgene portion ofthe DNA sequence of SEQ ID NO:8 and a similar length of 3′ flanking cornDNA of SEQ ID NO:6, for use as DNA primers in DNA amplification methodsare also disclosed in the present invention. Amplicons produced usingthese primers are diagnostic for corn event MON863. An amplicon producedby a first DNA primer homologous or complementary to SEQ ID NO:7 coupledwith a second DNA primer homologous or complementary to SEQ ID NO:5,when both are present together in a reaction mixture with corn eventMON863 DNA in a sample are an aspect of the present invention. Anamplicon produced by a third DNA primer homologous or complementary toSEQ ID NO:8 coupled with a fourth DNA primer homologous or complementaryto SEQ ID NO:6, when both are present together in a reaction mixturewith corn event MON863 DNA in a sample, are another aspect of thepresent invention. The corn plant MON863 and progeny derived therefromthat contain these DNA sequences used in a DNA amplification reaction toprovide one or more diagnostic amplicons are aspects of the invention.

According to yet another preferred embodiment of the present invention,methods of detecting the presence of a DNA corresponding to the cornevent MON863 event in a sample are provided. Such methods comprise thesteps of: (a) contacting a biological sample suspected of containing anevent MON863 DNA with a primer pair that, when used in a nucleic acidamplification reaction with said DNA, produces an amplicon that isdiagnostic for the corn event MON863; (b) performing a nucleic acidamplification reaction, thereby producing the amplicon; and (c)detecting the amplicon. The amplicons specifically exemplified hereincorrespond to a first amplicon of about 508 base pairs as set forth inSEQ ID NO:3 and a second amplicon of about 584 base pairs as set forthin SEQ ID NO:4, or longer or shorter amplicons, wherein said firstamplicon contains as least a nucleotide sequence corresponding to SEQ IDNO:1 from about nucleotide 1 through about nucleotide 11 or from aboutnucleotide 10 through about nucleotide 20 and said second ampliconcontains at least a nucleotide sequence corresponding to SEQ ID NO:2from about nucleotide 1 through about nucleotide 11 or from aboutnucleotide 10 through about nucleotide 20.

According to yet another preferred embodiment of the present invention,methods of detecting the presence of a DNA corresponding to the MON863event in a sample are provided. Such methods comprise the steps of: (a)contacting a biological sample suspected of containing an event MON863DNA with a probe that hybridizes under stringent hybridizationconditions with said DNA and that does not hybridize under stringenthybridization conditions with DNA from a control corn plant that doesnot contain an inserted DNA derived from pMON25097; (b) subjecting thesample and the probe to stringent hybridization conditions; and (c)detecting hybridization of the probe to the genomic DNA, whereindetection of probe binding to said DNA is diagnostic for the presence ofevent MON863 DNA in said sample.

According to a further preferred embodiment of the present invention,there is provided a novel corn plant MON863, that comprises DNAsequences comprising the novel transgene/genomic insertion regions asset forth in SEQ ID NO:3 and SEQ ID NO:4. The seeds of the plants ofMON863, the progeny of the plants of MON863 and the methods forproducing a corn plant by crossing the corn plant MON863 with itself orwith another corn plant are further embodiments of the presentinvention.

The foregoing and other preferred embodiments of the present inventionwill become more apparent from the following detailed descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES Description of Drawings

FIG. 1 illustrates a plant expression vector PV-ZMIR13, also designatedherein as pMON25097, from which the corn rootworm event MON863 isgenerated through particle acceleration technology using a Mlu Irestriction fragment from about nucleotide position 149 through aboutnucleotide position 4840.

FIG. 2 is a graphical map illustrating the general organization of theelements comprising the heterologous nucleic acid sequences insertedinto the corn event MON863 genome and essentially sets forth thepositions at which the inserted nucleic acid sequences are linked tocorn genomic DNA sequences designated herein as corn genomic nucleicacid sequences which flank the ends of the inserted heterologous DNAsequences; the corn event MON863 being characterized as follows: corngenomic DNA [1] flanking the arbitrarily assigned 5′ end of the fulllength primary functional inserted DNA sequence is adjacent to anon-naturally occurring CaMV35S AS4 promoter sequence [2] (P-CaMV.AS4,SEQ ID NO:17) operably connected to a wheat chlorophyll A/B bindingprotein untranslated leader sequence [3] (L-Ta.hcb1, SEQ ID NO:18)operably connected to a rice actin intron sequence [4] (I-Os.Act1, SEQID NO:19) operably connected to a non-naturally occurring sequenceencoding Cry3Bb variant protein [5] (SEQ ID NO:20) operably connected toa wheat heat shock Hsp17 transcription termination and polyadenylationsequence [6](T-Ta.Hsp17, SEQ ID NO:21), and the full-length primaryfunctional inserted DNA sequence being flanked by the corn genomic DNAat the arbitrarily assigned 3′ end [7], in which the junction between[1] and [2] ([8]) corresponds to SEQ ID NO:1, and the junction between[6] and [7] ([9]) corresponds to SEQ ID NO:2.

DESCRIPTION OF SEQUENCES

SEQ ID NO:1 corresponds to a junction sequence between corn genome andinserted DNA that is diagnostic for the arbitrarily assigned 5′ end ofthe full-length primary functional inserted DNA sequence in the cornevent MON863.

SEQ ID NO:2 corresponds to a junction sequence between corn genome andinserted DNA that is diagnostic for the arbitrarily assigned 3′ end ofthe full-length primary functional inserted DNA sequence in the cornevent MON863.

SEQ ID NO:3 corresponds to the sequences represented substantially by[1] and [2] of FIG. 2.

SEQ ID NO:4 corresponds to the sequences represented substantially by[6] and [7] of FIG. 2.

SEQ ID NO:5 corresponds to the partial corn genome DNA sequence that isadjacent to and flanking the 5′ end of the arbitrarily assigned 5′ endof the partial Cry3Bb DNA coding sequence inserted in the corn eventMON863.

SEQ ID NO:6 corresponds to the partial corn genome DNA sequence that isadjacent to and flanking the 3′ end of the arbitrarily assigned 3′ endof the partial Cry3Bb DNA coding sequence inserted in the corn eventMON863.

SEQ ID NO:7 corresponds to the sequence of the arbitrarily assigned 5′end of the partial Cry3Bb DNA coding sequence inserted in the corn eventMON863.

SEQ ID NO:8 corresponds to the sequence of the arbitrarily assigned 3′end of the partial Cry3Bb DNA coding sequence inserted in the corn eventMON863.

SEQ ID NO:9 corresponds to a 5′ primer sequence (primer A) complementaryto a part of the corn genomic DNA sequence identified as flanking thearbitrarily assigned 5′ end of the full length primary functionalinserted DNA sequence in the corn event MON863, and when paired with aprimer corresponding to the reverse complement of the sequence set forthin SEQ ID NO:10 and template DNA of the corn event MON863, produces anamplicon comprising SEQ ID NO:3 that is diagnostic for the corn eventMON863 DNA in a sample.

SEQ ID NO:10 corresponds to the reverse complement of a 3′ primersequence (primer B) complementary to a part of the arbitrarily assigned5′ end sequence of the full length primary functional DNA inserted intothe corn genome in the corn event MON863, and when paired with a primercorresponding to the sequence set forth in SEQ ID NO:9 and template DNAof the corn event MON863, produces an amplicon comprising SEQ ID NO:3that is diagnostic for the corn event MON863 DNA in a sample.

SEQ ID NO:11 corresponds to a 5′ primer sequence (primer C)complementary to part of the arbitrarily assigned 3′ end sequence of thefull length primary functional DNA inserted into the corn genome in thecorn event MON863, and when paired with a primer corresponding to thereverse complement of the sequence set forth in SEQ ID NO:12 andtemplate DNA of the corn event MON863, produces an amplicon having SEQID NO:4 that is diagnostic for the corn event MON863 DNA in a sample.

SEQ ID NO:12 corresponds to the reverse complement of a 3′ primersequence (primer D) complementary to a part of the corn genomic DNAsequence identified as flanking the arbitrarily assigned 3′ end of thefull length primary functional inserted DNA sequence in corn eventMON863, and when paired with a primer corresponding to the sequence setforth in SEQ ID NO:11 and the template DNA of the corn event MON863,produces an amplicon having SEQ ID NO:4 that is diagnostic for cornevent MON863 DNA in a sample.

SEQ ID NO:13 corresponds to a 5′ genome walker primer 1.

SEQ ID NO:14 corresponds to a 5′ genome walker primer 2.

SEQ ID NO:15 corresponds to a 3′ genome walker primer 1.

SEQ ID NO:16 corresponds to a 3′ genome walker primer 2.

SEQ ID NO:17 corresponds to CaMV35S AS4 promoter sequence.

SEQ ID NO:18 corresponds to a wheat chlorophyll A/B binding proteinuntranslated leader sequence (L-Ta.hcb1).

SEQ ID NO:19 corresponds to a rice actin intron sequence (I-Os.Act1).

SEQ ID NO:20 corresponds to a non-naturally occurring sequence encodinga Cry3Bb variant protein.

SEQ ID NO:21 corresponds to wheat heat shock Hsp17 transcriptiontermination and polyadenylation sequence (T-Ta.Hsp17).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994. The nomenclature forDNA bases as set forth at 37 CFR § 1.822 is used.

As used herein, the term “biological sample”, or “sample”, is intendedto include nucleic acids, polynucleotides, DNA, RNA, tRNA, cDNA, and thelike in a composition or fixed to a substrate which enables the sampleto be subjected to molecular probe analysis or thermal amplificationusing oligonucleotide probes and/or primers.

As used herein, the term “corn” means Zea mays or maize and includes allplant varieties that can be bred with corn, including wild maizespecies.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, the term “diagnostic” refers to the fact that, for thepurposes of identifying nucleic acid sequences as those contained withinor derived from the corn event MON863, any one or more of the novel DNAsequences set forth herein comprise the corn genome flanking sequencesadjacent to and linked to the arbitrarily assigned ends of the insertedheterologous DNA sequences are necessary and sufficient as beingdescriptive as a distinguishing characteristic of the corn event MON863genome, so long as the sequence comprises at least a part of one of theends of the inserted heterologous DNA sequence or the corn genomesequence flanking or adjacent to one of these ends and includes at leastthe two nucleotides, the di-nucleotide, comprising the point at whichthe corn genome sequence and the inserted heterologous DNA sequence arelinked together by a phosphodiester bond. It is well known in the artthat a sequence which is diagnostic for a particular event, such asthose disclosed herein for the corn event MON863, which is not presentin a particular sample containing corn genome nucleic acids, isindicative that the sample does not contain the diagnostic sequence andtherefore the nucleic acids in the sample are not or were not derivedfrom and have not been contained within the genome of the corn eventMON863. In addition, additional novel and diagnostic sequences arepresent within the corn event MON863 DNA as exemplified herein selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, andSEQ ID NO:4 and complements thereof.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety thatinclude the heterologous DNA. Even after repeated backcrossing to arecurrent parent, the inserted DNA and flanking DNA from the transformedparent is present in the progeny of the cross at the same chromosomallocation. The term “event” also refers to DNA from the originaltransformant comprising the inserted DNA and flanking genomic sequenceimmediately adjacent to the inserted DNA that would be expected to betransferred to a progeny that receives inserted DNA including thetransgene of interest as the result of a sexual cross of one parentalline that includes the inserted DNA (e.g., the original transformant andprogeny resulting from selfing) and a parental line that does notcontain the inserted DNA.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two or moreindependently segregating exogenous genes (exogenous genes referringnucleotide sequences that are not naturally occurring in the plantgenome, i.e., heterogeneous to the corn plant). Selfing of appropriateprogeny can produce plants that are homozygous for any combination ofthe exogenous genes. Backcrossing to a parental plant and out-crossingwith a non-transgenic plant are also contemplated, as is vegetativepropagation. Descriptions of other breeding methods that are commonlyused for different traits and crops can be found in one of severalreferences, e.g., Fehr, in Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

A “probe” is an isolated nucleic acid to which a conventional detectablelabel or reporter molecule, e.g., a radioactive isotope, ligand,chemiluminescent agent, or enzyme may be linked or attached. Such aprobe is complementary to a sequence within a target nucleic acid, inthe case of the present invention, to a sequence of genomic DNA from thecorn event MON863 whether from a corn plant or from a sample thatincludes DNA from the event. Probes according to the present inventioninclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that bind specifically to a targetDNA sequence and can be used to detect the presence of that target DNAsequence.

“Primers” are isolated nucleic acid probes that are annealed to, for anygiven single primer, a complementary target DNA sequence by nucleic acidhybridization to form a hybrid between the primer and the target DNAsequence, and then extended along the target DNA strand by a polymerase,e.g., a DNA polymerase. Primer pairs of the present invention refer totwo or more different primer sequences for is in amplification of anucleic acid sequence that is between and linked to the target sequencesdesignated as the reverse complement or substantially the reversecomplement of the primers, e.g., by the polymerase chain reaction (PCR)or other conventional nucleic-acid amplification methods.

Probes and primers are generally from about 11 nucleotides or more inlength, preferably from about 18 nucleotides or more in length, morepreferably from about 24 nucleotides or more in length, and mostpreferably from about 30 nucleotides or more in length. Such probes andprimers hybridize specifically to a target sequence under highstringency hybridization conditions. Preferably, probes and primersaccording to the present invention have complete sequence similaritywith the target sequence, although probes differing from the targetsequence and that retain the ability to hybridize to target sequencesmay be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR-primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as Primer(Version 0.5, © 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes constructed based on the flanking DNA, insertsequences, and junction sequences disclosed herein can be used toconfirm the presence of the disclosed sequences in a sample byconventional methods, e.g., by recloning and sequencing such sequences.

Any single nucleic acid probe or primer of the present inventionhybridizes under stringent conditions to a specific target DNA sequence.Any conventional nucleic acid hybridization or amplification method canbe used to identify the presence of DNA from a transgenic event in asample. Nucleic acid molecules or fragments thereof specificallyhybridize to other nucleic acid molecules under certain circumstances.As used herein, two different nucleic acid molecules each comprisingdifferent sequences, are said to specifically hybridize to one anotherif the two molecules form an anti-parallel, double-stranded nucleic acidstructure. A nucleic acid molecule is said to be the “complement” ofanother nucleic acid molecule if they exhibit complete complementarity.As used herein, molecules are said to exhibit “complete complementarity”when every nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989, and by Haymes et al.(In: Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C., 1985). Departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure. In order for a nucleic acid molecule to serve as a primer orprobe it needs only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence, and that thehybridization is detectable.

As used herein, an “isolated” nucleic acid is one that has beensubstantially separated or purified away from other nucleic acidsequences in the cell of the organism in which the nucleic acidnaturally occurs, i.e., other chromosomal and extrachromosomal DNA andRNA, by conventional nucleic acid-purification methods. The term alsoembraces recombinant nucleic acids and chemically synthesized nucleicacids.

As used herein, a “substantially homologous” sequence is a nucleic acidsequence that specifically hybridizes to the complement of the nucleicacid sequence to which it is being compared, i.e., the target sequence,under high stringency conditions. Appropriate stringency conditionswhich promote DNA hybridization, for example, 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C., are known to those skilled in the art or can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.,6.3.1-6.3.6., 1989. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or either the temperature orthe salt concentration may be held constant while the other variable ischanged. In a preferred embodiment, a nucleic acid of the presentinvention will specifically hybridize to one or more of the nucleic acidmolecules set forth either in SEQ ID NO:1 or SEQ ID NO:2 or complementsthereof or fragments of either under moderately stringent conditions,for example at about 2.0×SSC and about 65° C. In a particularlypreferred embodiment, a nucleic acid of the present invention willspecifically hybridize to one or more of the nucleic acid molecules setforth either in SEQ ID NO:1 or SEQ ID NO:2 or complements or fragmentsof either under high stringency conditions. A nucleic acid of thepresent invention that hybridizes to a nucleic acid sequence comprisingSEQ ID NO:1 or to a nucleic acid sequence comprising SEQ ID NO:3 willnot necessarily hybridize to a nucleic acid sequence comprising SEQ IDNO:2 or to a nucleic acid sequence comprising SEQ ID NO:4, and viceversa.

In one aspect of the present invention, a preferred marker nucleic acidmolecule of the present invention has the nucleic acid sequence setforth in SEQ ID NO:1 or in SEQ ID NO:2 or complements thereof orfragments of either. In another aspect of the present invention, apreferred marker nucleic acid molecule of the present invention sharesbetween 80% and 100% or between 90% and 100% sequence identity with thenucleic acid sequence set forth in SEQ ID NO:1 and SEQ ID NO:2 orcomplement thereof or fragments of either. In a further aspect of thepresent invention, a preferred marker nucleic acid molecule of thepresent invention shares between 95% and 100% sequence identity with thesequence set forth in SEQ ID NO:1 and SEQ ID NO:2 or complement thereofor fragments of either. SEQ ID NO:1 and SEQ ID NO:2 may be used asmarkers in plant breeding methods to identify the progeny of geneticcrosses similar to the methods described for simple sequence repeat DNAmarker analysis, in “DNA markers: Protocols, Applications, andOverviews, 173-185, Cregan, et al., eds., Wiley-Liss NY, 1997. Thehybridization of the probe to the target DNA molecule can be detected byany number of methods known to those skilled in the art, these caninclude, but are not limited to, fluorescent tags, radioactive tags,antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the individual primers in aprimer pair to hybridize only to the individual and unique targetnucleic-acid sequence to which each primer, comprising the correspondingwild-type sequence (or its complement), would bind, and preferably toproduce a unique amplification product, the amplicon, in a DNA thermalamplification reaction.

As used herein, the term “transformation” refers to the transfer of anucleic acid fragment into the genome of a host organism such as a hostplant, resulting in genetically stable inheritance. Host plantscontaining the transformed nucleic acid fragments are referred to as“transgenic plants”.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic-acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thecorn plant resulting from a sexual cross contains transgenic eventgenomic DNA from the corn plant MON863 of the present invention, DNAextracted from a corn plant tissue sample may be subjected to a nucleicacid amplification method using a primer pair that includes a primerderived from the flanking sequence in the genome of the plant adjacentto the insertion site of the inserted heterologous DNA, and a secondprimer derived from the inserted heterologous DNA to produce an ampliconthat is diagnostic for the presence of the event DNA. The amplicon is ofa length and has a sequence that is also diagnostic for the event. Theamplicon may range in length from the combined length of the primerpairs plus one nucleotide base pair, preferably plus about fiftynucleotide base pairs, more preferably plus about two hundred-fiftynucleotide base pairs, and even more preferably plus about fourhundred-fifty nucleotide base pairs. Alternatively, a primer pair can bederived from the flanking sequence on both sides of the inserted DNA soas to produce an amplicon that includes the entire insert nucleotidesequence. A member of a primer pair derived from the plant genomicsequence may be located in a distance from the inserted DNA sequence,this distance can range from one nucleotide base pair up to about twentythousand nucleotide base pairs. The use of the term “amplicon”specifically excludes primer dimers that may be formed in the DNAthermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic Press, San Diego, 1990. PCRamplification methods have been developed to amplify up to 22 kb ofgenomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc.Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as othermethods known in the art of DNA amplification may be used in thepractice of the present invention. The sequence of the heterologous DNAinsert or the flanking sequence from the corn event MON863 can beverified (and corrected if necessary) by amplifying such sequences fromthe event using primers derived from the sequences provided hereinfollowed by standard DNA sequencing of the PCR amplicon or of the clonedDNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide isdesigned which overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microwell plate. Following PCR of the region of interest(using one primer in the inserted sequence and one in the adjacentflanking genomic sequence), a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. DNTPs are added individually and theincorporation results in a light signal that is measured. The lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen, et al., (Genome Res. 9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedwhich overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. A single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14: 303-308, 1996).Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking genomic and insert DNA junction. The unique structure of theFRET probe results in it containing secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Thefluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization, andis diagnostic for the corn event MON863 nucleic acid in a sample.

All of the above methods can be modified to determine the zygosity of aparticular sample of nucleic acids derived from a single source. Forexample, a corn event MON863 plant which is homozygous for the event 863allele contains within its genome two copies of the event 863 allelecharacteristic of and diagnostic for the corn event MON863 genome, andthus when selfed would breed true. Alternatively, a corn event MON863homozygous plant can be crossed with another variety of corn, and theresult of that cross would be plants that were heterozygous for theevent MON863 allele. Methods are envisioned in which one skilled in theart could determine the zygosity of a particular plant with reference tothe event MON863 allele.

For example, the use of three different primers in an amplificationreaction with corn event MON863 DNA as a template, and in a separate andparallel amplification reaction with negative control corn DNA that isnot MON863, i.e., that does not contain the inserted DNA present withinMON863 DNA, would result in two different outcomes depending on thezygosity of the corn DNA containing the corn event MON863 DNA. Exemplaryprimers could be selected from the group consisting of SEQ ID NO:9, SEQID NO:10, and SEQ ID NO:12. Amplification of non-MON863 DNA with thisgroup of primers would result in primer pair SEQ ID NO:10 and SEQ IDNO:12 producing a first amplicon corresponding to the contiguous corngenome sequence into which the PV-ZMIR13 sequence was inserted, thatamplified sequence corresponding substantially to the linked combinationof SEQ ID NO:5 and SEQ ID NO:6. This first amplicon would be expected ina plant that was heterozygous for the corn event MON863 allele, however,a heterozygote would also produce a second amplicon corresponding to SEQID NO:3 from the extension of the primer pair corresponding to SEQ IDNO:9 and SEQ ID NO:10. A corn plant containing DNA that was homozygousfor the MON863 allele would only produce the second amplicon.

Similarly, a third amplicon would be produced from a thermalamplification reaction that used the primers SEQ ID NO:10, SEQ ID NO:11,and SEQ ID NO:12 with template DNA from a MON863 corn plant, this thirdamplicon corresponding to SEQ ID NO:4. This third amplicon would be theonly amplicon produced using this particular combination of primers andtemplate DNA if the plant was homozygous for the MON863 allele, however,heterozygote template DNA would result in the amplification of the firstand the third amplicons, and non-MON863 template DNA would result in theamplification of only the first amplicon.

Herein, the inventors have determined as judged by molecularcharacterization that corn event MON863 contains a primary functionalinsert containing a significant portion of the transformation plasmid,PV-ZMIR13. This segment is detectable and diagnostic for the eventMON863 nucleic acid sequences in a sample, in particular in plants thathave been selfed since the origination of the MON863 event.

There are many methods for transforming the Cry3Bb nucleic acidmolecules into plant cells such as maize plant cells to produce adesired event such as MON863. Suitable methods are believed to includevirtually any methods by which nucleic acid molecules may be introducedinto the cells, such as by Agrobacterium infection or direct delivery ofnucleic acid molecules that may include PEG-mediated transformation,electroporation and acceleration of DNA coated particles, etc.(Pottykus, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:205-225, 1991;Vasil, Plant Mol. Biol. 25: 925-937, 1994). For example, electroporationhas been used to transform Zea mays protoplasts (Fromm et al., Nature312:791-793, 1986). In general, the following are four most commonlyused general methods for delivering a gene into cells: (1) chemicalmethods (Graham and van der Eb, Virology, 54:536-539, 1973); (2)physical methods such as microinjection (Capecchi, Cell 22:479-488,1980), electroporation (Wong and Neumann, Biochem. Biophys. Res. Commun.107:584-587, 1982; Fromm et al., Proc. Natl. Acad. Sci. (USA)82:5824-5828, 1985; U.S. Pat. No. 5,384,253); and the gene gun (Johnstonand Tang, Methods Cell Biol. 43:353-365, 1994); (3) viral vectors(Clapp, Clin. Perinatol. 20:155-168, 1993; Lu et al., J. Exp. Med.178:2089-2096, 1993; Eglitis and Anderson, Biotechniques 6:608-614,1988); and (4) receptor-mediated mechanisms (Curiel et al., Hum. Gen.Ther. 3: 147-154, 1992; Wagner et al., Proc. Natl. Acad. Sci. (USA) 89:6099-6103, 1992).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. See for example(Potrykus et al., Mol. Gen. Genet., 205:193-200, 1986; Lorz et al., Mol.Gen. Genet., 199:178, 1985; Fromm et al., Nature, 319:791, 1986;Uchimiya et al., Mol. Gen. Genet.:204:204, 1986; Callis et al., Genesand Development, 1183, 1987; Marcotte et al., Nature, 335:454, 1988).Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Among them are the methods for corn (U.S. Pat. No. 5,569,834, U.S. Pat.No. 5,416,011; McCabe et al., Biotechnology 6:923, 1988; Christou etal., Plant Physiol., 87:671-674, 1988). Illustrative methods for theregeneration of cereals from protoplasts are also described (Fujimura etal., Plant Tissue Culture Letters, 2:74, 1985; Toriyama et al., Theor.Appl. Genet. 205:34, 1986; Yamada et al., Plant Cell Rep. 4: 85, 1986;Abdullah et al., Biotechnology, 4:1087, 1986).

A transgenic plant such as a transgenic corn MON863 plant formed usingtransformation methods typically contains a single added Cry3Bb gene onone chromosome. Such a transgenic plant can be referred to as beingheterozygous for the added Cry3Bb gene. More preferred is a transgenicplant that is homozygous for the added Cry3Bb gene; i.e., a transgenicplant that contains two added Cry3Bb genes, one gene at the same locuson each chromosome of a chromosome pair. A homozygous transgenic plantcan be obtained by sexually mating (selfing) an independent segregatedtransgenic plant that contains a single added Cry3Bb gene, germinatingsome of the seeds produced and analyzing the resulting plants producedfor the Cry3Bb gene.

It is understood that two different transgenic plants can also be matedto produce offspring that contain two independently segregating addedCry3Bb genes. Selfing of appropriate progeny can produce plants that arehomozygous for both added Cry3Bb genes that encode Cry3Bb polypeptides.Backcrossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation.

Specifically, a method for producing a corn plant that is resistant tocoleopteran insect infestation may be conducted with the followingsteps: 1) sexually crossing a first corn plant grown from the corn seedevent MON863 comprising a DNA molecule selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 andSEQ ID NO:20 that confers resistance to coleopteran insect infestation,and a second corn plant that lacks the resistance to coleopteran insectinfestation, thereby producing a plurality of first progeny plants; 2)selecting a first progeny plant that is resistant to coleopteran insectinfestation; 3) selfing said first progeny plant, thereby producing aplurality of second progeny plants; and 4) selecting from said secondprogeny plants a plant resistant to coleopteran insect infestation. Thefirst progeny plant that is resistant to coleopteran insect infestationor the second progeny plant that is resistant to coleopteran insectinfestation may be backcrossed to the second corn plant or a third cornplant resulting in a corn plant that is resistant to coleopteran insectdamage infestation.

The regeneration, development, and cultivation of plants such as theMON863 plants from transformants or from various transformed explantsare well known in the art (Weissbach and Weissbach, In: Methods forPlant Molecular Biology, Eds., Academic Press, Inc. San Diego, Calif.,1988). This regeneration and growth process may typically include thesteps of selection of transformed cells containing exogenous Cry3Bbgenes, culturing those individualized cells through the usual stages ofembryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil.

The regeneration of plants containing the foreign, exogenous gene thatencodes a protein of interest is well known in the art. As described inthe present invention, the regenerated plants such as the regeneratedMON863 plants that contain the Cry3Bb nucleic acids, either wild type orchemically synthesized, that encode for the Cry3Bb proteins, may bepreferably self-pollinated to provide homozygous transgenic maizeplants, as discussed before. Otherwise, pollen obtained from theregenerated maize plants may be crossed to seed-grown plants ofagronomically important lines. Conversely, pollen from plants of theseimportant lines is used to pollinate regenerated plants. A transgenicMON863 plant of the present invention may be cultivated using methodswell known to one skilled in the art.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. Transformation of monocot plants using electroporation,particle bombardment, and Agrobacterium has also been reported.Transformation and plant regeneration have been achieved in many monocotplants that include maize, asparagus, barley and wheat, etc. (Bytebieret al., Proc. Natl. Acad. Sci. USA 84:5345, 1987; Wan and Lemaux, PlantPhysiol 104:37, 1994; Rhodes et al., Science 240: 204, 1988; Gordon-Kammet al., Plant Cell, 2:603, 1990; Fromm et al., Bio/Technology 8:833,1990; Armstrong et al., Crop Science 35:550-557, 1995; Vasil et al.,Bio/Technology 10:667, 1992; U.S. Pat. No. 5,631,152).

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, 1989; Mailga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press, 1995;Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor,N.Y., 1997).

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for identification of corn event MON863 DNA in a sample andcan be applied to methods for breeding corn plants containing the MON863DNA. The kits contain one or more DNA sequences comprising at least 11contiguous nucleotides homologous or complementary to sequences selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, and complements thereof. These DNAsequences can be used in DNA amplification reactions or as probes in aDNA hybridization method.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1 Isolation and Characterization of the DNA SequencesFlanking the MON863 Insertion Event

Corn event MON863 was generated through particle acceleration technologyusing a 4.7-Kb agarose gel-isolated Mlu I restriction fragment from theplasmid vector PV-ZMIR13 (pMON25097, FIG. 1). The plant expressionvector pMON25097 contains a first expression cassette comprising anon-naturally occurring CaMV35S AS4 promoter sequence (P-CaMV.AS4, SEQID NO:17) operably connected to a wheat chlorophyll A/B binding proteinuntranslated leader sequence (L-Ta.hcb1, SEQ ID NO:18) operablyconnected to a rice actin intron sequence (I-Os.Act1, SEQ ID NO:19)operably connected to a non-naturally occurring sequence encoding Cry3Bbvariant protein (SEQ ID NO:20) operably connected to a wheat heat shockHsp17 transcription termination and polyadenylation sequence(T-Ta.Hsp17, SEQ ID NO:21). The plant expression vector pMON25097contains a second expression cassette linked to the Cry3Bb expressioncassette that confers paromomycin resistance to transformed plant tissue(i.e. the 3′ end of the cry3Bb expression cassette is linked to the 5′end of the second expression cassette conferring paromomycinresistance). This resistance cassette consists of an enhanced CaMV35Spromoter sequence (U.S. Pat. No. 5,164,316) that is operably connectedto a neomycin phosphotransferase coding sequence (U.S. Pat. No.5,569,834) that is operably connected to a nopaline synthasetranscription termination and polyadenylation sequence (Fraley et al.Proc. Natl. Acad. Sci. USA 80:4803-4807, 1983). Transgenic corn plantsresistant to paromomycin were derived essentially as described in U.S.Pat. No. 5,424,412.

Molecular characterization of the insert in the corn event MON863demonstrated that one copy of the DNA fragment used for transformationis present in the corn event MON863. In order to develop event-specificPCR identification methods, the sequences of corn DNA flanking the 5′and 3′ ends of the insert in the corn event MON863 were determined usingGenomeWalker™ technology (Clontech Laboratories, Inc.) in accordancewith the manufacturer's instructions. The GenomeWalker™ method involvesfirst completely digesting purified corn MON863 DNA with differentrestriction enzymes provided in the GenomeWalker™ kit that leave bluntends. Next, the purified blunt-ended genomic DNA fragments are ligatedto GenomeWalker™ Adaptors comprising known nucleic acid fragments. Eachligation is then amplified in a first PCR reaction using an outeradaptor primer, SEQ ID NO:22 (5′-GTAATACGACTCACTATAGGGC-3′) provided byGenomeWalker™ and an outer, gene-specific primer (SEQ ID NO:13,5′-GAACGTCTTCTTTTTCCACGATGCTCC-3′, and SEQ ID NO:15,5′-GCGAGTCTGATGAGACATCTCTGTAT-3′, for the 5′ and 3′ ends of thetransgene insert, respectively). The first PCR product mixture is thendiluted and used as a template for a secondary or nested PCR with thenested adaptor primer, SEQ ID NO:23 (5′-ACTATAGGGCACGCGTGGT-3′) providedby GenomeWalker™ and a nested gene-specific primer (SEQ ID NO:14,5′-TCGGCAGAGGCATCTTGAATGATAGC-3′, and SEQ ID NO:16,5′-AATTTGGTTGATGTGTGTGCGAGTTCT-3′, for the 5′ and 3′ ends of thetransgene insert, respectively). The secondary PCR product, which beginswith the known gene-specific sequences and extends into the unknownadjacent genomic DNA, can then be sequenced using methods well known inthe art. Once the flanking corn genomic sequences were determined, PCRassays capable of detecting the presence of corn plant PV-ZMIR13(MON863) DNA in a sample were developed.

Following this procedure, the nucleotide sequence as set forth in SEQ IDNO:5 was characterized as the corn genome sequence that is immediatelyadjacent to and upstream of the arbitrarily assigned 5′ end of thepMON25097 DNA fragment that was inserted into the corn genome resultingin the construction and isolation of transgenic corn event MON863. Oneskilled in the art, or even one of ordinary skill in the art, wouldrealize that additional nucleotide sequence information can readily beobtained that is even more distal from the junction sequence as setforth in SEQ ID NO:1 but still within the corn genome than the present242 nucleotides exemplified herein in SEQ ID NO:5, and from nucleotideposition 267 through nucleotide position 508 as set forth in SEQ IDNO:3. Also, the nucleotide sequence as set forth in SEQ ID NO:6 wascharacterized as the corn genome sequence that is immediately adjacentto and downstream of the arbitrarily assigned 3′ end of the pMON25097DNA fragment that was inserted into the corn genome resulting in theconstruction and isolation of transgenic corn event MON863. One skilledin the art will also realize that additional nucleotide sequenceinformation can readily be obtained that is even more distal from thejunction sequence as set forth in SEQ ID NO:2 but still within the corngenome than the present 224 nucleotides exemplified herein in SEQ IDNO:6, and from nucleotide position 361 through nucleotide position 584as set forth in SEQ ID NO:4.

Example 2 Detection of the Presence of MON863 DNA in a Sample

The following provides a non-limiting example of the PCR assaysdeveloped to detect the presence of the MON863 DNA in a sample.

DNA was extracted from approximately 100 mg of ground grain tissue usingQiagen's Dneasy Plant Mini Kit (catalog # 68163, Valencia, Calif.)according to the manufacturer's recommended protocol with one exception.The grain used was processed prior to extraction in a −80° C. freezer,and not ground under liquid nitrogen using a mortar and pestleimmediately prior to extraction. DNA quantitation was conducted usingmethods well-known in the art, a Hoefer DNA Quant 200 Fluorometer, andBoehringer Mannheim (Indianapolis, Ind.) molecular size marker IX as aDNA calibration standard.

PCR analysis of the genomic DNA sequences flanking the 5′ end of theinsert in MON863 was performed using one primer (primer A) derived fromthe 5′ genomic flanking sequence (SEQ ID NO:9,5′-GTCTTGCGAAGGATAGTGGGAT-3′) paired with a second primer (primer B)located near the 5′ end of the inserted DNA in the 35S promoter (SEQ IDNO:10, 5′-CATATGACATAAGCGCTCTTGG-3′), covering a 508-bp region. The PCRanalysis for genomic DNA sequences flanking the 3′ end of the MON863insert was conducted using one primer (primer D) derived from the 3′genomic flanking sequence (SEQ ID NO:12, 5′-AGACTCTATGCTCTGCTCATAT-3′)paired with a second primer (primer C) located in the tahsp17polyadenylation sequence near the 3′ end of the insert spanning a 584-bpregion (SEQ ID NO:11, 5′-CTGATCATTGGTGCTGAGTCCTT-3′) (FIG. 2). The PCRanalyses were conducted using 50 ng of the corn event MON863 genomic DNAor a MON846 non-transgenic genomic DNA template in a 50 μL reactionvolume containing a final concentration of 1.5 mM Mg²⁺, 0.4 μM of eachprimer, 200 μM each dNTP, and 2.5 units of Taq DNA polymerase. Thereactions were performed under the following cycling conditions: 1 cycleat 94° C. for 3 minutes; 38 cycles of 94° C. for 30 seconds, 60° C. for30 seconds, 72° C. for 1.5 minutes; 1 cycle at 72° C. for 10 minutes.

The PCR products (20 μL) of the expected sizes representing the genomicsequence flanking the 5′ and 3′ ends of the insert were isolated by gelelectrophoresis on a 2.0% agarose gel at 60 V for ˜1 hour and visualizedby ethidium bromide staining. The PCR fragments representing the 5′ and3′ flanking sequences were excised from the gel and purified using theQIAquick Gel Extraction Kit (Qiagen, catalog # 28704) following theprocedure supplied by the manufacturer. The purified PCR products werethen sequenced with the initial PCR primers using dye-terminatorchemistry.

The control reactions containing no template as well as the reactionscontaining non-transgenic corn DNA did not generate a PCR product witheither primer set, as expected. PCR analysis of the corn rootworm eventMON863 DNA generated the expected size products of 508 bp representingthe 5′ flanking sequence (SEQ ID NO:3) when using primers A and B havingSEQ ID NOs: 9 and 10 and 584 bp representing the 3′ flanking sequence(SEQ ID NO:4) when using primers D and C having SEQ ID NOs: 11 and 12.

Sequence data indicated that the 5′ amplicon, i.e., SEQ ID NO:3,consisted of 266 bp of the 5′ end of the 35S promoter at the 5′ end ofthe insert followed by 242 bp of corn genomic flanking DNA. Sequencedata indicated that the 3′ amplicon, i.e., SEQ ID NO:4, consisted of 360bp of the tahsp17 3′ polyadenylation sequence which defines the 3′ endof the insert, immediately followed by 224 bp of corn genomic flankingDNA.

Agronomically and commercially important products and/or compositions ofmatter including but not limited to animal feed, commodities, and cornproducts and by-products that are intended for use as food for humanconsumption or for use in compositions that are intended for humanconsumption including but not limited to corn flour, corn meal, cornsyrup, corn oil, corn starch, popcorn, corn cakes, cereals containingcorn and corn by-products, and the like are intended to be within thescope of the present invention if these products and compositions ofmatter contain detectable amounts of the nucleotide sequences set forthherein as being diagnostic for the corn event MON863.

Seed comprising the MON863 corn event have been deposited by theApplicant with American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va., USA ZIP 20110-2209 on Oct. 17, 2000. The ATCCprovided the Applicant with a deposit receipt, assigning the ATCCDeposit Accession No. PTA-2605 to the corn Zea mays event MON863PV-ZMIR13.

Those of skill in the art, in light of these examples, should appreciatethat many changes can be made to the foregoing assays to detect DNAderived from corn event MON863 in a sample. For example, a primer setcomprising one primer complementary to corn genome DNA and anotherprimer complementary to sequences within the insert are envisioned.Furthermore, any of various hybridization assays described earlier usingDNA probes complementary to the novel nucleic acid sequences located attransgene/genome junctions are envisioned as well.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1. A biological sample derived from corn event MON863 plant, tissue, orseed, wherein said sample comprises a nucleotide sequence which is or iscomplementary to a sequence selected from the group consisting of SEQ IDNO:1 and SEQ ID NO:2, wherein said nucleotide sequence is detectable insaid sample using a nucleic acid amplification or nucleic acidhybridization method and, wherein a representative sample of said cornevent MON863 seed of has been deposited with American Type CultureCollection (ATCC) with Accession No. PTA-2605.
 2. The biological sampleof claim 1, wherein said biological sample comprises plant, tissue, orseed of transgenic corn event MON863.
 3. The biological sample of claim2, wherein said biological sample is a DNA sample extracted from thetransgenic corn plant event MON863, and wherein said DNA samplecomprises one or more of the nucleotide sequences selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, and the complementthereof.
 4. The biological sample of claim 3, wherein said biologicalsample is selected from the group consisting of corn flour, corn meal,corn syrup, corn oil, corn starch, and cereals manufactured in whole orin part to contain corn by-products.
 5. An extract derived from cornevent MON863 plant, tissue, or seed and comprising a nucleotide sequencewhich is or is complementary to a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO:2, wherein a representativesample of said corn event MON863 seed of has been deposited withAmerican Type Culture Collection (ATCC) with Accession No. PTA-2605. 6.The extract of claim 5, wherein said nucleotide sequence is detectablein said extract using a nucleic acid amplification or nucleic acidhybridization method.
 7. The extract of claim 6, wherein said extractcomprises plant, tissue, or seed of transgenic corn plant event MON863.8. The extract of claim 7, further comprising a composition selectedfrom the group consisting of corn flour, corn meal, corn syrup, cornoil, corn starch, and cereals manufactured in whole or in part tocontain corn by-products, wherein said composition comprises adetectable amount of said nucleotide sequence.
 9. A corn event MON863,wherein a representative sample of seed of said corn event has beendeposited with American Type Culture Collection (ATCC) with AccessionNo. PTA-2605.
 10. Plant parts of the corn event of claim
 9. 11. Seedcomprising corn event MON863, wherein said seed comprises a DNA moleculeselected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:2, andwherein a representative sample of corn event MON863 seed of has beendeposited with American Type Culture Collection (ATCC) with AccessionNo. PTA-2605.